1. Field of the Invention
The present invention relates generally to systems and methods designed to monitor the relative concentrations of different isotopic forms of a chemical species in a sample, and more specifically to systems and methods designed to monitor the relative concentration of .sup.13 CO.sub.2 and .sup.12 CO.sub.2 in human breath.
2. Discussion of the Related Art
Helicobacter pylori (H.pylori) is an organism that colonizes the mucous gel layer of the gastric antrum. This bacterium is believed to be responsible for more than twenty million cases of chronic gastritis in the United States, implicating H. Pylori as the major cause of non-autoimmune chronic gastritis. In addition, scientific data indicate that this organism may be the predominant cause of more than five million cases of peptic ulcer disease in the United States. Furthermore, prospective epidemiologic studies indicate that H. pylori may play a role in the pathogenesis of gastric cancer and lymphoma. In particular, the presence of this organism in children is suspected of producing a significant risk for the development of cancer in adulthood. Moreover, since H. Pylori is estimated to be present in about one billion people worldwide, it may be the most common human pathogen. Although at present eradication of H. Pylori is only recommended for patients with H.pylori infection and peptic ulcer disease, more wide-scale eradication programs aimed at decreasing the incidence of gastric malignancies are under consideration.
As is generally true of any test method, it is desirable to monitor the presence of H. Pylori using a minimally invasive technique. Based on its lack of invasiveness, the .sup.13 C-urea breath test is commonly believed to be the best approach to monitoring the presence of H.pylori in humans. In this test, breath samples are obtained from a patient before and after ingestion of a meal containing .sup.13 C-urea. The carbon dioxide present in a breath sample is then analyzed to determine the ratio of .sup.13 CO.sub.2 to .sup.12 CO.sub.2. Typically, the isotope ratio measurement is made from about 20 minutes to about 60 minutes after ingestion of the .sup.13 C-urea.
When performing a .sup.13 C-urea breath test, the criteria for a positive test response for H. pylori is a .sup.13 CO.sub.2 to .sup.12 CO.sub.2 ratio increase of 5 to 6 parts per thousand above the baseline ratio measured prior to ingestion of the .sup.13 C-urea. The normal baseline isotopic abundance of .sup.13 C is about 1% while .sup.12 C is about 99%. The combination of the low initial concentration of .sup.13 CO.sub.2 and the small change in its concentration for the minimum positive response to the .sup.13 C-urea test requires a measurement technique with very high sensitivity to both .sup.13 CO.sub.2 and .sup.12 CO.sub.2, and their ratio.
Due to their high sensitivity and good signal to noise ratio, magnetic sector ratio mass spectrometers are normally employed in .sup.13 C-urea breath tests. According to this technique, the relative isotope concentrations of carbon dioxide are calculated by monitoring the size of the spectral peaks at mass numbers 44 (.sup.12 CO.sub.2) and 45 (.sup.13 CO.sub.2). However, the current technique has several substantial drawbacks. The mass spectrometers are relatively expensive and so are only available in a small number of specialized analytical laboratories. In addition, portions of the vacuum systems in these spectrometers require bake-out cycles on a daily basis to remove contamination, and the filaments in the ionization source typically require weekly replacement. Hence, the current apparatus has a relatively low duty cycle. Moreover, the current method also has problems with interference from .sup.17 O, which must be separately monitored, adding to the complexity of the measurement. Furthermore, the current technique requires a sample transfer step from the sample container to a vacuum system, increasing the possibility of sample contamination. Because of these factors, highly-skilled technicians are commonly used for sample preparation as well as operation and maintenance of the instrumentation. As a result, magnetic sector mass spectrometry is a relatively expensive and inconvenient analytical method for conducting .sup.13 C-urea breath tests.
Infrared (IR) absorption has also been used to measure .sup.13 CO.sub.2 /.sup.12 CO.sub.2 ratios in breath samples. In IR absorption techniques, a chemical species undergoes a transition between vibrational energy states by absorbing electromagnetic radiation at a wavelength that corresponds to the energy difference between two vibrational energy levels of the chemical species. Since the difference in vibrational energy levels for a given chemical species (e.g., carbon dioxide) depends upon the mass of the chemical species, absorption of IR radiation by different isotopic forms of the chemical species (e.g., .sup.13 CO.sub.2 and .sup.12 CO.sub.2) can be distinguished by their respective IR absorption bands which appear at different wavelengths.
A method of performing .sup.13 C-urea breath tests by high resolution infrared absorption spectroscopy with a continuously tunable semiconductor diode laser as the source of electromagnetic radiation is disclosed in Applied Optics 32, 6727 (1993). However, as is generally true for any laser-based analytical technique, this method involves relatively expensive instrumentation and careful temperature control to avoid problems associated with laser output power and wavelength instability. Moreover, due to the relatively weak absorption of carbon dioxide at a wavelength of about 1.6 microns, this system employs a comparatively long absorption pathlength (23.6 meters), accomplished with a multiple pass cell. Furthermore, the cells have a relatively large volume of from about 300 cm.sup.3 to about 400 cm.sup.3, so more than one breath may be required to fill and flush the cells with a breath sample, reducing the practicality of this technique for breath analysis. In addition misalignment of cell mirrors due to mechanical vibrations can lead to substantial errors in the isotope ratio measurement, increasing the likelihood of unreliable results.
U.S. Pat. No. 5,394,236 and Science 263, 945 (1994) each disclose methods of conducting .sup.13 C-urea breath tests with a discretely tunable CO.sub.2 gas laser. However, these techniques have the inherent stability problems of any laser-based technique as discussed above. In addition, the wavelength of the electromagnetic radiation output by the lasers is used at two different emission lines. Therefore, the systems utilize either two separate lasers or a sophisticated switching mechanism, either of which lead to increased system cost. Moreover, to avoid the relatively weak signals and long path lengths associated with transitions from the ground vibrational state of dilute concentrations of carbon dioxide, the disclosed systems in these references analyze plasma conductivity with optogalvanic effect spectroscopy. According to these methods, the breath sample is disposed within a plasma discharge to place carbon dioxide in excited vibrational energy states. A change in the conductivity of the plasma discharge due to transitions from the excited vibrational energy states is used to determine the relative concentrations of .sup.13 CO.sub.2 and .sup.12 CO.sub.2. While optogalvanic effect spectroscopy may avoid certain disadvantages of other analytical methods, the use of a plasma discharge results in a system having increased complexity and cost.
The Lancet 345, 961 (1995), U.S. Pat. No. 5,486,699 and Anal. Chem. 58, 2172 (1986) each disclose methods of using IR absorption to monitor the relative concentrations of .sup.13 CO.sub.2 and .sup.12 CO.sub.2 in a gas sample with non-dispersive IR sources. The first reference does not disclose the specific details of this technique, but the second and third references do disclose that a comparatively large sample volume (at least 500 mL) is needed. Therefore, more than one breath may be required to fill and flush the cells, reducing the practicality of this technique for breath analysis. The last reference discloses a method that involves modulating the pressure or density of a gas sample contained in four different sample cells in order to obtain good sensitivity. As a result, this technique is relatively complex and expensive to utilize.
Gastroenterology 108 (4 Suppl.), A103 (1995) discloses a system for measuring H. Pylori in humans with an IR spectrometer. The figure demonstrates fine resolution of the rotational energy transitions, indicating that a high spectral resolution was used; these high resolution spectrometers are relatively large and inconvenient and comparatively expensive to purchase. Furthermore, such spectrometers often involve relatively long data acquisition times when operated with high spectral resolution.
Gastroenterology 108 (4 Suppl.), A235 (1995) discloses the results of measurements of measuring H. Pylori in humans using an IR spectrometer. This reference does not disclose the experimental details of the method used, nor the specific apparatus used.
In addition to the .sup.13 C-urea breath test, a related .sup.14 C-urea breath test is available which takes advantage of the radioactive decay of .sup.14 C to monitor the presence of H. Pylori in humans. The detection instrumentation for this test is based on counting radioactive decay particles and, therefore, is relatively simple and inexpensive. However, the use of a radioactive material involves substantial costs associated with safe material handling and disposal protocol. Moreover, the use of a radioactive element precludes this test from being used in children and pregnant women. This limitation is a major disadvantage since a primary target for proposed H.pylori eradication and testing programs is children.
Therefore, there is a need for a safe, simple, effective, low-maintenance and inexpensive system and method for monitoring the relative concentrations of isotopic and non-isotopic forms of a chemical species.